Modulation of gene expression in mammalian cells by small duplex RNAs is typically associated with recognition of mRNA (Siomi and Siomi, 2009). Duplex RNAs complementary to gene promoters have been reported to either silence or activate gene expression in mammalian cells (Morris et al., 2004; Ting et al., 2005; Janowski et al., 2005; Li et al., 2006; janowski et al., 2007). Argonaute 2 (AGO2), a key protein involved in RNAi (Liu et al., 2004), is required for the action of promoter-targeted RNAs5,8, and a related protein, AGO1, has also been implicated in the mechanism (Kim et al., 2006). Recent reports have suggested that the mechanism of promoter-targeted RNAs involves recognition of noncoding transcripts that overlap gene promoters (Han et al., 2007; Schwartz et al., 2008). Over 70% of all genes have noncoding transcripts that overlap their promoters and these transcripts provide potential target sites for small RNA duplexes (He et al., 2008; Kapranov et al., 2007; Sun et al., 2005; Gingeras, 2007; Wahlstedt, 2006; Amaral and Mattick 2008)).
Promoter-targeted RNAs are robust modulators of progesterone receptor (PR) transcription in T47D and MCF7 breast cancer cells (Janowski et al., 2005; Janowski et al., 2007; Janowski et al., 2006; Schwartz et al., 2008). The inventors term these small RNAs antigene RNAs (agRNAs) to distinguish them from duplex RNAs that target mRNA. The main difference between activation or inhibition of gene expression by closely related agRNAs is the basal expression of PR. Gene silencing is observed in T47D cells that constitutively express PR at high basal levels, while activation of PR expression is observed in MCF7 cells that express PR at low levels (Janowski et al., 2007).
Both activating and inhibitory agRNAs modulate PR expression through binding to complementary target sequences within an antisense transcript that originates from inside the PR gene and is transcribed through the promoter region. agRNAs recruit AGO protein to the antisense transcript, affect levels of RNA polymerase II (RNAP2) at the promoter, and alter the mix of regulatory proteins that bind the antisense transcript and the PR promoter (Schwartz et al., 2008).
Noncoding RNAs also overlap the 3′-untranslated region (3′-UTR) of many genes (Gingeras, 2007; wahlstedt, 2006; Amaral and Mattick, 2008). The 3′-UTR plays a major role in cellular regulation and disease pathology (Chen et al., 2006) and is involved in a variety of post-transcriptional processes, including mRNA transport, localization, and stability. The function of 3′ noncoding transcripts is unclear, but their proximity to the 3′-UTR suggests that they may affect gene regulation.
The abundance of transcripts that overlap the 3′-UTR, coupled with the ability of agRNAs to modulate gene expression by targeting overlapping 5′ transcripts, suggested that small RNAs might also influence gene expression by recognizing sequences beyond the 3′ end of genes. However, there has been little investigation into the potential function of overlapping noncoding transcripts at the 3′-region of genes, and no examination of whether these noncoding transcripts might be targets for modulating gene expression by duplex RNAs.